Abstract:

Post-translational modifications of proteins by phosphorylation and methylation are employed to transduce information in living organisms. Enteric bacteria such as Escherichia coli and Salmonella typhimurium utilize a combination of these methods to regulate their motility via the bacterial chemotaxis system. Chemotactic proteins are activated using phosphorylation, while adaptation is achieved through dephosphorylation and methylation. When the response regulator, CheY, is phosphorylated at a conserved aspartate residue, it favors the occurrence of a tumbling event and a subsequent change in the organism's direction of swimming. It is critical that the system recover so that normal swimming is restored. To promote this, CheY has a cognate phosphatase, CheZ. CheY also has a slower intrinsic autophosphatase activity. This dissertation establishes that the predominant mechanism of dephosphorylation of CheY at physiological pH and temperature involves hydrolysis at the phosphorus, rather than attack on the carbonyl carbon of the aspartate, regardless of whether CheZ is present or not. However, evidence is presented that an intramolecular dephosphorylation reaction can occur for phospho-CheY under some conditions. This intramolecular route may occur via a cyclic succinimide intermediate by a mechanism analogous to the deamidation reaction observed for aging peptides and proteins. In support of this hypothesis, a mutant CheY was purified in which the conserved aspartate 57 is changed to an asparagine (D57N). This species was shown to deamidate at rates much faster than predicted based on primary structure alone. Analogous mutants in other proteins of the Haloacid Dehalogenase (HAD) superfamily, of which CheY is a member, also exhibit this type of catalyzed deamidation. Thus, the active site of CheY and other members of the HAD superfamily may have evolved to catalyze nucleophilic substitution chemistry.